Role of pyruvate dehydrogenase and insulin in the regulation of lipogenesis in the lactating mammary gland of the rat during the starved-refed transition.

Administration of insulin with glucose to starved lactating rats, which activates pyruvate dehydrogenase [M. A. Baxter & H. G. Coore (1978) Biochiem. J. 174, 553-561], restored lipogenesis in mammary gland in vivo to 50% of the value observed in refed (2.5 h) rats. The correlations between pyruvate dehydrogenase activity and the rate of lipogenesis persisted in isolated acini. Activation of pyruvate dehydrogenase in vitro with dichloroacetate increased lipogenesis from [6-14C]glucose in acini from starved and refed rats by 250% and 100% respectively. However, in the presence of dichloroacetate, only 70% of the increased flux through pyruvate dehydrogenase was converted into lipid in acini from starved rats, whereas all of the increase could be accounted for as lipid in acini from refed rats. Addition of insulin plus dichloroacetate was required to obtain maximal rates of lipogenesis in acini from starved rats. Similarly, insulin increased the incorporation of [1-14C]acetate into lipid only in acini from starved rats. Although the activity of pyruvate dehydrogenase plays an important role in the control of mammary-gland lipogenesis, the evidence presented suggests a second regulatory site which is insulin-sensitive and is located after the generation of cytosolic acetyl-CoA.     

Modulation of intracellular cyclic AMP content and rate of lipogenesis in mammary acini in vitro.

Relationships between the cyclic AMP content, the rate of lipogenesis and the activity of acetyl-CoA carboxylase in acini prepared from lactating rat mammary tissue were investigated by exposing them to agents that increase their cyclic AMP content in the presence or absence of insulin. The dose-dependent inhibition of lipogenesis by theophylline in acini isolated from fed rats was highly correlated with the induced increases in acinar cyclic AMP content. Cyclic AMP of acini from 24 h-starved lactating rats was more sensitive in its response to theophylline than that in acini from fed animals. Neither forskolin nor a mixture of isoprenaline and Ro 7-2956 were able significantly to change either the rate of lipogenesis or the activity of acetyl-CoA carboxylase in acini from fed rats when added to incubations in vitro, in spite of the large increases in cyclic AMP concentration produced by these agents. Insulin was without effect on the activity of acetyl-CoA carboxylase and on either the basal or isoprenaline-stimulated cyclic AMP content of acini. These results are discussed in terms of the possibility that the rate of lipogenesis and the cyclic AMP content in mammary acini can vary independently of one another and of the activity of acetyl-CoA carboxylase.     

Development of insulin sensitivity in white adipose tissue during the suckling-weaning transition in the rat. Involvement of glucose transport and lipogenesis.

The changes of insulin responsiveness of white adipose tissue during the suckling-weaning transition in the rat were investigated in vitro on isolated adipocytes. Insulin binding, glucose transport and glucose metabolism in adipocytes from suckling rats and from rats weaned on to a high-carbohydrate (HC) or a high-fat (HF) diet were compared. Despite similar insulin binding, insulin-stimulated glucose transport rate is lower in adipocytes from suckling rats and HF-weaned rats than in adipocytes from HC-weaned rats. Moreover, whereas insulin markedly stimulates glucose metabolism in adipocytes from HC-weaned rats, glucose metabolism is totally unresponsive to insulin in adipocytes from suckling and HF-weaned rats. This insulin resistance is associated with a very low rate of lipogenesis and low activities of acetyl-CoA carboxylase, fatty acid synthase and pyruvate dehydrogenase.     

Stimulation of hepatic lipogenesis and acetyl-coenzyme A carboxylase by vasopressin.

The effect of vasopressin on the short-term regulation of fatty acid synthesis was studied in isolated hepatocytes from rats fed ad libitum. Vasopressin stimulates fatty acid synthesis by 30-110%. This increase is comparable with that obtained with insulin. Angiotensin also stimulates fatty acid synthesis, whereas phenylephrine does not. The dose-response curve for vasopressin-stimulated lipogenesis is similar to the dose-response curve for glycogenolysis and release of lactate plus pyruvate. Vasopression also stimulates acetyl-CoA carboxylase activity in a dose-dependent manner. Vasopressin does not relieve glucagon-inhibited lipogenesis, whereas insulin does. The action of vasopressin on hepatic lipogenesis is decreased, but not suppressed, in Ca2+-depleted hepatocytes. The results suggest that vasopressin acts on lipogenesis by increasing availability of lipogenic substrate (lactate + pyruvate) and by activating acetyl-CoA carboxylase.     

Control of glucose metabolism in isolated acini of the lactating mammary gland of the rat. The ability of glycerol to mimic some of the effects of insulin.

Inhibition of glucose uptake by acetoacetate and relief of this inhibition by insulin found previously in slices of rat mammary gland [Williamson, McKeown & Ilic (1975) Biochem. J. 150. 145-152] was confirmed in acini, which represent a more homogeneous population of cells. Glycerol (1mM) behaved like insulin (50 minuits/ml) in its ability to relieve the inhibition of glucose (5 mM) utilization caused by acetoacetate (2 mM) in acini. Both glycerol and insulin reversed the increase in [citrate] and the decrease in [glycerol 3-phosphate] and the [lactate]/[pyruvate] ratio in the presence of acetoacetate. Lipogenesis from 3H2O, [3-14C] acetoacetate, [1-14C]- and [6-14C]-glucose was stimulated, whereas 14CO2 formation from [3-14C]acetoacetate was decreased. Neither insulin nor glycerol relieved the acetoacetate inhibition of glucose uptake when lipogenesis was inhibited by 5-(tetradecyloxy)-2-furoic acid. From measurements of [3-14C]acetoacetate incorporation into lipid in the various situations it is suggested that a cytosolic pathway for acetoacetate utilization may exist in rat mammary gland. In the absence of acetoacetate, glycerol inhibited glucose utilization by 60% and increased both [glycerol 3-phosphate] and the [lactate/[pyruvate] ratio. Possible ways in which glycerol may mimic the effects of insulin are discussed.     

Effects of inhibition of protein synthesis by cycloheximide on lipogenesis in mammary gland and liver of lactating rats.

1. Administration of cycloheximide (an inhibitor of protein synthesis) to lactating rats raised the concentrations of amino acids, and in particular, the branched-chain amino acids (valine, leucine and isoleucine) in blood, liver and mammary gland. 2. Inhibition of protein synthesis increased the incorporation in vivo of L-[U-14C]leucine into lipids of mammary gland and liver. 3. Cycloheximide treatment caused no immediate change in the overall rate of lipogenesis in vivo (measured with 3H2O) in mammary gland but increased the rate in liver 3-fold; this latter effect also occurred in livers of virgin rats. 4. The increased rate of hepatic lipogenesis was not accompanied by significant changes in the plasma insulin concentration or the activity of acetyl-CoA carboxylase. 5. Although cycloheximide decreased the entry of total triacylglycerol into the circulation it did not alter the rate of secretion of newly synthesized saponifiable lipid. 6. Cycloheximide slightly stimulated lipogenesis from endogenous substrates in isolated hepatocytes, but this effect was abolished when lactate was the exogenous substrate. 7. Administration of cycloheximide to virgin rats decreased liver glycogen and increased the hepatic content of glucose 6-phosphate, pyruvate and lactate. 8. It is concluded that (a) there is no short-term link between the rate of protein synthesis and lipogenesis in the lactating mammary gland and (b) the increased rate of hepatic lipogenesis in cycloheximide-treated rats is mainly due to stimulation of glycogenolysis, glycolytic flux and consequent increased availability of pyruvate.     

Modulation of the activity of insulin-dependent enzymes of lipogenesis by glucocorticoids.

Administration of triamcinolone or dexamethasone to rats led to a prompt, marked and persistent rise in liver acetyl-CoA carboxylase activity. The activity of fatty acid synthetase increased to a lesser extent and after a more prolonged glucocorticoid treatment, whereas the changes in that of NADP-malate dehydrogenase and ATP-citrate lyase were not appreciable. The overall channeling of [1-14-C]acetyl-CoA to fatty acids was enhanced. The triamcinolone effect on acetyl-CoA carboxylase activity appeared to be dependent on the coincident hyperinsulinemia since it was not obtained in alloxan-diabetic rats, whereas the alanine-aminotransferase-inducing effect of this hormone was additive to that of insulin deficiency. In adipose tissue triamcinolone treatment caused a reduction in the activity of all lipogenesis enzymes and blunted their response to insulin administration. The antagonism of glucocorticoids toward insulin, selectively modulating the responses of the insulin-sensitive enzymes in liver and adipose tissue is discussed. The rise in hepatic lipogenic capacity, through the retention of the ability of insulin to induce acetyl-CoA carboxylase, may be physiologically important in restraining the ketogenesis from acetyl-CoA despite the increased fat utilization during glucocorticoid excess.     

Impaired lipogenesis in mammary glands of lactating rats fed on a cafeteria diet. Reversal of inhibition of glucose metabolism in vitro by insulin.

In lactating rats fed on a cafeteria diet (chow plus palatable high-energy foods) the decreased glucose uptake and lipogenesis in vitro in acini correlated with the depressed mammary-gland lipogenesis in vivo. Insulin in vitro restored the rate of glucose uptake and its conversion to lipid to values approaching those for acini from rats fed on the chow diet alone.     

Effect of insulin on ketogenesis and fatty acid synthesis in rat hepatocytes incubated with dichloroacetate

In parenchymal liver cells isolated from fed rats, insulin increased the formation of 14CO2 from [1-14C]pyruvate (and presumably the flux through pyruvate dehydrogenase) by 14%. Dichloroacetate, an activator of the pyruvate dehydrogenase complex, stimulated this process by 133%. As judged from the conversion of [2-14C]pyruvate to 14CO2, the tricarboxylic acid cycle activity was not affected by insulin, but it was depressed by dichloroacetate. In hepatocytes from fed rats, incubated with glucose as the only carbon source, dichloroacetate caused a stimulation (31%) of fatty acid synthesis, measured as 3H incorporation from 3H2O into fatty acid, and an increased (134%) accumulation of ketone bodies (acetoacetate + D-3-hydroxybutyrate). Dichloroacetate did not affect ketone body formation from [14C]palmitate, suggesting that the increased accumulation of ketone bodies resulted from acetyl-CoA derived from pyruvate. Insulin stimulated fatty acid synthesis in hepatocytes from fed rats. In the combined presence of insulin plus dichloroacetate, fatty acid synthesis was more rapid than in the presence of either insulin or dichloroacetate, whereas the accumulation of ketone bodies was smaller than in the presence of dichloroacetate alone. Although pyruvate dehydrogenase activity, which is rate-limiting for fatty acid synthesis in hepatocytes from fed rats, is stimulated both by insulin and by dichloroacetate, the reciprocal changes in fatty acid synthesis and ketone body accumulation brought about by insulin in the presence of dichloroacetate suggest that insulin is also involved in the regulation of fatty acid synthesis at a mitochondrial site after pyruvate dehydrogenase, possibly at the partitioning of acetyl-CoA between citrate and ketone body formation.     

Effect of hypophysectomy and insulin on lipogenesis in cockerels.

Hypophysectomy increases hepatic lipogenesis in cockerels. In an attempt to find the possible hormonal mechanism for this we have examined the effects of hypophysectomy, insulin and pituitary homogenates on hepatic lipogenesis. Insulin (5 U/kg) injected intravenously, simultaneously with glucose-14C tracer, increased the amount of 14C found in liver lipids at 30 min after the injection. Similarly, insulin injected 5 min before killing, increased the incorporation of glucose-14C and acetate-14C by liver slices during a 30 min incubation. Hypophysectomy increased lipogenesis within 24 hours. The effect of insulin was most pronounced in hypophysectomized cockerels. The activity of the lipogenic enzyme, acetyl CoA carboxylase was similarly affected. A homogenate of chicken pituitaries added to the medium reduced lipid synthesis from glucose-14C by liver slices. This effect was larger in liver slices in which lipogenesis had been stimulated by insulin. The increased rate of hepatic lipgenesis in hypophysectomized cockerels may be caused partly by increased hepatic sensitivity to the lipogenic action of insulin or by the removal of a direct inhibition by pituitary hormones.     

Both insulin and epidermal growth factor stimulate lipogenesis and acetyl-CoA carboxylase activity in isolated adipocytes. Importance of homogenization procedure in avoiding artefacts in acetyl-CoA carboxylase assay.

Epidermal growth factor (EGF) stimulates lipogenesis by 3-4-fold in isolated adipocytes, with a half-maximal effect at 10 nM-EGF. In the same batches of cells insulin stimulated lipogenesis by 15-fold. Freezing and prolonged homogenization of adipocytes results in release of large quantities of pyruvate carboxylase from broken mitochondria, and sufficient pyruvate can be carried through into assays for this enzyme to cause significant interference with assays of acetyl-CoA carboxylase in crude adipocyte extracts. This may account for the high amount of citrate-independent acetyl-CoA carboxylase activity reported to be present in adipocyte extracts in some previous publications. This problem may be eliminated by homogenizing very briefly without freezing. By using the modified homogenization procedure, EGF treatment of adipocytes was shown to produce an effect on acetyl-CoA carboxylase activity almost identical with that of insulin. Both messengers increase Vmax. without significant effect on the Ka for the allosteric activator, citrate.     

Proline and hepatic lipogenesis

The effects of proline on lipogenesis in isolated rat hepatocytes were determined and compared with those of lactate, an established lipogenic precursor. Proline or lactate plus pyruvate increased lipogenesis (measured with 3H2O) in hepatocytes from fed rats depleted of glycogen in vitro and in hepatocytes from starved rats. Lactate plus pyruvate but not proline increased lipogenesis in hepatocytes from starved rats. ( - )-Hydroxycitrate, an inhibitor of ATP-citrate lyase, partially inhibited incorporation into saponifiable fatty acid of 3H from 3H2O and 14C from [U-14C]lactate with hepatocytes from fed rats. Incorporation of 14C from [U-14C]proline was completely inhibited. Similar complete inhibition of incorporation of 14C from [U-14C]proline by ( - )-hydroxycitrate was observed with glycogen-depleted hepatocytes or hepatocytes from starved rats. Inhibition of phosphoenolpyruvate carboxykinase by 3-mercaptopicolinate did not inhibit the incorporation into saponifiable fatty acid of 3H from 3H2O or 14C from [U-14C]proline or [U-14C]lactate. Both 3-mercaptopicolinate and ( - )-hydroxycitrate increased lipogenesis (measured with 3H2O) in the absence or presence of lactate or proline with hepatocytes from starved rats. The results are discussed with reference to the roles of phosphoenolpyruvate carboxykinase, mitochondrial citrate efflux, ATP-citrate lyase and acetyl-CoA carboxylase in proline- or lactate-stimulated lipogenesis.     

The role of rapid lipogenesis in insulin secretion: Insulin secretagogues acutely alter lipid composition of INS-1 832/13 cells

Pancreatic beta cell mitochondria convert insulin secretagogues into products that support insulin exocytosis. We explored the idea that lipids are some of these products formed from acyl group transfer out of mitochondria to the cytosol, the site of lipid synthesis. There are two isoforms of acetyl-CoA carboxylase, the enzyme that forms malonyl-CoA from which C₂ units for lipid synthesis are formed. We found that ACC1, the isoform seen in lipogenic tissues, is the only isoform present in human and rat pancreatic islets and INS-1 832/13 cells. Inhibitors of ACC and fatty acid synthase inhibited insulin release in islets and INS-1 cells. Carbon from glucose and pyruvate were rapidly incorporated into many lipid classes in INS-1 cells. Glucose and other insulin secretagogues acutely increased many lipids with C₁₄-C₂₄ chains including individual cholesterol esters, phospholipids and fatty acids. Many phosphatidylcholines and phosphatidylserines were increased and many phosphatidylinositols and several phosphatidylethanolamines were decreased. The results suggest that lipid remodeling and rapid lipogenesis from secretagogue carbon support insulin secretion.     

Insulin stimulation of glucose transport and metabolism in a human Wilms' tumor-derived myoblast-like cell Line: Modulation of hormone effects by glucose deprivation

The effects of insulin and glucose on parameters of metabolism were investigated in myoblast-like (MBL) cells, a human myoblast-like cell line derived from a Wilms' tumor. Insulin responses were studied after 4 hr pre-incubation in serum free media, with or without 5 mM glucose. Insulin was added during the last 2 hr. Glucose starvation markedly increased basal glucose transport (measured as 2-deoxyglucose uptake) as well as the net uptake of [¹⁴C]glucose and [¹⁴C]glucose incorporation into glycogen. Insulin stimulated net glucose uptake and incorporation into glycogen in a dose-dependent manner in glucose-fed and starved cells. These insulin responses were markedly enhanced in glucose-starved cells. Insulin accelerated 2-deoxyglucose transport in glucose-fed cells but did not further stimulate basal glucose transport in glucose-deprived cells. Insulin increased the incorporation of [³H]leucine into protein in glucose-fed or -starved MBL cells equally. The dose of insulin required for half-maximal insulin responses was similar for all parameters studied. Cycloheximide did not prevent the increased basal glucose incorporation in glucose-starved cells, but markedly inhibited the insulin response, while in glucose-fed cells, cycloheximide stimulated basal glucose incorporation. We conclude that MBL cells resemble fibroblasts in their insulin-independent stimulation of glucose transport in response to glucose-deprivation; when provided with glucose, they respond to insulin like fibroblasts. However, after brief glucose-starvation, the stimulated glucose transport system is no longer insulin-responsive in MBL cells, while pathways leading to the synthesis of macromolecules demonstrate preserved or enhanced stimulation by insulin, suggesting that these cells may serve as models to study the regulation of receptor-response coupling by the metabolic milieu.     

The regulation of lipogenesis in vivo in the lactating mammary gland of the rat during the starved-refed transition. Studies wtih acarbose, a glucosidase inhibitor.

Depression of carbohydrate digestion by oral administration of acarbose, a glucosidase inhibitor, led to a 75% inhibition of the re-activation of lipogenesis in vivo in the mammary gland of 18 h-starved lactating rats refed with 5 g of chow diet. Rates of [1-14C]glucose incorporation in vitro into lipid and CO2 in mammary-gland acini isolated from refed animals were elevated compared with acini from starved rats, but acarbose treatment completely prevented this stimulation. Gastric intubation of glucose led to a large stimulation of lipogenesis in the mammary gland of starved lactating rats, similar to that induced by refeeding with chow diet; this was dependent on the amount of glucose given and the time elapsed between glucose administration and injection of 3H2O for the measurement of lipogenesis. The switch-on of lipogenesis in the mammary gland of starved lactating rats, by refeeding or by intubation of glucose, was associated with a decrease in the ratio of [glucose 6-phosphate]/[fructose 1,6-bisphosphate] in the gland, indicative of an increase in phosphofructokinase activity. A time-course study revealed that the ratio decreased rapidly over the first 30 min of chow refeeding, after which a large surge in lipogenesis was seen. Acarbose, given 25 min after the onset of refeeding, led to a stepwise increase in the ratio, in parallel with the observed decrease in lipogenic activity. It is concluded that the control of lipogenesis in the mammary gland is closely linked to the availability of dietary carbohydrate. An important site of regulation of lipogenesis in the gland appears to be at the level of phosphofructokinase. A possible role of insulin in the regulation of phosphofructokinase activity, and the acute modulation of insulin-sensitivity in the gland during the starved-refed transition, are discussed.     

Adrenaline responsiveness of glucose metabolism in insulin-resistant adipose tissue of rats fed a high-fat diet.

The effects of adrenaline (0.5 microM) and the combination of adrenaline and insulin (1.7nM) on [6-14C]glucose metabolism were assessed in epididymal fat-pads from rats fed either a low- or high-fat diet. The response of lipolysis to adrenaline was clearly diminished in fat-fed rats. Insulin added to adrenaline inhibited the lipolysis by 50% regardless of the diet. Glucose utilization in adipose tissue of fat-fed rats was markedly stimulated by adrenaline (glucose uptake was increased 3-fold and the production of CO2 and the glycerol moiety of acylglycerol was increased 4-fold). However, adipose tissue from fat-fed rats was resistant to the effect of insulin to produce a further increase in adrenaline-stimulated glucose uptake. The intracellular capacity of lipogenesis on the one hand, and the production of CO2 and the glycerol moiety of acylglycerol on the other, are of prime importance in the action of insulin and adrenaline on glucose utilization in this model.     

Effects of fasting and refeeding on the in vitro insulin sensitivity of rat aorta

Rat aorta responds to refeeding after a fast in a manner similar to adopose tissue and liver by developing an enhanced capacity for lipogenesis and glycogen synthesis from glucose. The in vitro incorporation of D-U-14C-glucose into aortic triglycerides and glycogen was two- to four-fold higher in rats refed for three to five days after a three day fast than in ad libitum fed controls. Insulin significantly stimulated this incorporation only during refeeding for three days after a three-day fast. The glycogen synthesizing system appeared to be stimulated and to become sensitive to insulin earlier in the refeeding process than did the lipogenic system. The in vitro incorporation of 14C-glucose into aortic phospholipids was less affected by the nutritional state of the animal, and was not stimulated by insulin at any stage of the experiment. Possible mechanisms for the development of insulin supersensitivity and the implications for lipid accumulation in the artery wall are discussed.     

Some aspects of metabolic adaptations in lipid metabolism during starvation are mimicked by epinephrine in rat adipocytes

1. The effects of fasting on the neutral lipid synthesis to insulin and/or epinephrine in isolated fat cells have been examined using [1-14C]glucose. 2. The ability of adipocytes from starved rats to synthesize fatty acids from both labeled substrates was markedly diminished compared to adipocytes from control rats. 3. The response of lipogenic stimulation to insulin at all concentrations tested was greatly diminished in adipocytes from 24 hr starved rats. 4. [1-14C]glucose utilization rates in the absence or in the presence of insulin were not significantly different in adipocytes from 24 hr starved rats as compared with control adipocytes, although basal and insulin stimulated glyceride-glycerol synthesis were significantly higher in starved adipocytes. 5. Epinephrine acutely inhibited [1-14C]acetate incorporation into fatty acids for insulin-stimulated lipogenesis in control adipocytes, in contrast, this lipolytic agent strongly increased [1-14C]glucose conversion to triacylglycerols. 6. In both cases, the differences in lipid synthesis capacities found in both nutritional states were abolished by epinephrine.     

Rapid inhibition by intragastric triolein of the re-activation of glucose utilization and lipogenesis in the mammary gland during the starved-refed transition in lactating rats. Evidence for a direct effect of oral lipid on mammary tissue.

1. Oral administration of triacylglycerol (triolein) to starved/chow-refed lactating rats suppressed the lipogenic switch-on in the mammary gland in vivo. 2. A time-course study revealed that triolein, administered at 30 min after the onset of refeeding, had no influence on lipogenic rate in the mammary gland between 30 and 60 min, but markedly decreased it between 60 and 90 min. Glucose uptake by the mammary gland (arteriovenous difference) increased by 30 min of refeeding, as did lactate production. Between 30 and 90 min glucose uptake remained high in the control animals, but glucose uptake and net C3-unit uptake were decreased in the triolein-loaded animals by 90 min. 3. Triolein increased [glucose 6-phosphate] in the gland and simultaneously decreased [fructose 1,6-bisphosphate], indicative of a decrease in phosphofructokinase activity. This cross-over occurred at 60 min, i.e. immediately before the inhibition of lipogenesis, and by 90 min had reached 'starved' values. 4. Triolein had no effect on plasma [insulin] nor on whole-blood [glucose], [lactate] or [3-hydroxybutyrate]; a small increase in [acetoacetate] was observed. 5. Infusion of the lipoprotein lipase inhibitor, Triton WR1339, abolished the suppression of mammary-gland lipogenesis by triolein and the increase in the [glucose 6-phosphate]/[fructose 1,6-bisphosphate] ratio, suggesting a direct influence of dietary lipid on mammary-gland glucose utilization and phosphofructokinase activity.